Islamic University Gaza Faculty of Health Sciences Medical

Islamic University_ Gaza Faculty of Health Sciences. Medical Technology Department. Biochemistry lab. MEDI 2130 Qualitative amino acid tests

Objectives: • General information about amino acids. • Qualitative tests of amino acids.

Introduction Food are divided into three classes : 1 - Carbohydrate Source of energy. 2 - Lipid Principal of energy reserve. 3 - Proteins Energy for growth and cellular maintance.

Amino acid structure (Building blocks of proteins which linked to peptide bond ) Each amino acid consists of : 1. Central carbon atoms 2. An amino acid 3. Carboxyl group 4. Side chain (All amino acids found in proteins have this basic structure, differing only in the structure of the Rgroup or the side chain. )

Classification of amino acids according source • Essential amino acids: Humans incapable of forming requisite and must be Required in diet. • Non essential amino acids: Not required in diet.

• The simplest, and smallest, amino acid found in proteins is glycine for which the R-group is a hydrogen (H). • Proline It is unique among the 20 proteinforming amino acids in that the amine nitrogen is bound to not one but two alkyl groups, thus making it a secondary amine. • There are 20 natural amino acids that are found within proteins. All of them are L-α amino acids. ( except glycine, is achiral carbon) because it has four different groups bonded to it. ).

Classification of amino acids according to their (polarity) in water 1 - Non-polar (Hydrophobic amino acid) : are amino acid that contain C, H in their side chain (hate water, normally buried inside the protein core) 2 - Uncharged polar. 3 -polar amino acids: amino acid that contain in their side chain O, N and they can dissolve in water ( like dissolve like ) hydrophilic (love water), tend to found on surface A-Basic polar (positively charged). B- Acidic polar (negatively charged).

Note : Polar amino acids are more soluble in water than non-polar.

At acidic p. H, the carboxyl group is protonated and the amino acid is in the cationic form At neutral p. H, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions At alkaline p. H, the amino group is neutral –NH 2 and the amino acid is in the anionic form.

v Iso electric point (PI) : It is the p. H value at which concentration of anionic and cationic groups are equal (i. e. the net charge of this molecule equals zero). Each amino acid have a different PI

Qualitative test for amino acids • There number of test to detect the presence of amino acid • This is largely depend on the natural of side chain

Amino acids analysis A. Solubility test. B. The presence of proteins in a solution is often detected by general tests, such as biuret or specific tests that depend on the presence of a specific amino acid. 1. Ninhydrin test: for α-L amino acids 2. Xanthoproteic test: for Aromatic amino acids 3. Lead sulfite test: detection of amino acids containing sulfhydral group (- SH)

4. Millon's test: for amino acids containing hydroxy phenyl group 5. Sakaguchi Test. 6. Hopkins-Cole (Glyoxylic Acid Reaction) 7. biuret test

Solubility test. • Objective: To investigate the solubility of selected amino acid in various solutions -Principle: • Polar amino acids are soluble in polar solvent, and vice versa. • The solubilities of amino acids in water are highly variable.

• The solubility of amino acids and proteins is largely dependent on the solution p. H. • The structural changes in an amino acid or protein that take place at different p. H values alter the relative solubility of the molecule. • In acidic solutions, both amino and carboxylic groups are protonated. • In basic solutions, both groups are deprotonated. Amino acids are essentially soluble in water. Their solubilities in water, dilute alkali and dilute acid vary from one compound to the other depending on the structure of their side chains.

Solubility of Amino Acids • You will already have dissolved a number of amino acids in water for the previous experiments. In this test try to dissolve tyrosine and cystine in water and note the result. Procedure • Put 2. 0 m. L (34 drops) of water in a clean test tube. Take a small portion of the contents of the vial marked “tyrosine” on a spatula and add it to the water. Shake well. Note if the tyrosine has dissolved. If it has not, then heat the test tube and note if the tyrosine dissolves. Is tyrosine readily soluble/sparingly soluble/insoluble in water? Repeat this procedure with cystine.

Ninhydrin Principle: • 1. Ninhydrin (triketohydrindene hydrate) degrades amino acids into aldehydes (on p. H range 4), ammonia and CO 2 though a series of reactions. • 2. The reducon product obtained from ninhydrin (hydrindantin) then reacts with NH 3 and excess ninhydrin to produce an intensely blue or purple pigment, sometimes called ruhemann's purple(diketohydrin). • This reaction provides an extremely sensitive test for amino acids. • alpha-amino acid + 2 ninhydrin ---> CO 2 + aldehyde + final complex(Bl. UE) + 3 H 2 O

• The imino acids proline and hydroxyproline also react with ninhydrin, but they give a yellow colored complex instead of a purple one. • Besides amino acids, other complex structures such as peptides, peptones and proteins also react positively when subjected to the ninhydrin reaction. Ninhydrin_ethanol reagent is flammable. Toxic, and irritant. Keep away from Bunsen burner flames prevent eye, skin, clothing contact. Avoid inhaling the vapors or ingesting the reagent.

With all amino acid will give purple or deep blue with exception Proline gives yellow not violet (why)? The secondary amino group (imino group) of proline residues is held in rigid conformation that reduces the structural flixibility of polypeptide regions containing proline. Proline does not give ninhydrin reaction as this reagent requires free alpha amino group but proline have imino group

• Procedure • To 1 m. L solution add 5 drops of 0. 5% ninhydrine solution • Boil over a water bath for 2 min. • Allow to cool and observe the blue color formed. Results:

Xanthoproteic test : Objective: • to differentiate between aromatic amino acids which give positive results [yellow color] and other amino acids. Principle: • Concentrated nitric acid react with aromatic nucleus present in the amino acid side chain [nitration reaction] giving the solution yellow color. Note: • Amino acids tyrosine and tryptophan contain activated benzene rings [aromatic nucleus] which are easily nitrated to yellow colored compounds. • The aromatic ring of phenyl alanine dose not react readily with nitric acid despite it contains a benzene ring, but it is not activated, therefore it will not react

Procedure • To 2 m. L amino acid solution in a boiling test tube, add equal volume of concentrated HNO 3. • Heat over a flame for 2 min and observe the color. • Now COOL THOROUGHLY and CAUTIOSLY run in sufficient 3 ml Na. OH (why) • Observe the color of the nitro derivative of aromatic nucleus.

Hopkins-Cole (Glyoxylic Acid Reaction) Aim: Specific for tryptophan (the only amino acid containing indole group) Principle: • Reacting with a glyoxylic acid in the presence of a strong acid, the indole ring forms a violet cyclic product. • The protein solution is hydrolyzed by conc. H 2 SO 4 at the solution interface. • Once the tryptophan is free, it reacts with glyoxylic acid to form violet product.

Procedure 1. In a test tube, add to 2 ml of the solution an equal volume of Hopkins- Cole reagent and mix thoroughly. • Incline the tube and let 5 to 6 ml of conc. H 2 S 04 acid flow slowly down the side of the test tube, thus forming a reddish - violet ring at the interface of the two layers. That indicates the presence of tryptophan

4. Millon's test v Objective: • This test is specific for tyrosine. Because it is the only amino acid containing a phenol group. • Note: phenol group, a hydroxyl group attached to benzene ring. Millon’s reagent contains mercury and HNO 3 and is very toxic, corrosive a strong oxidant, an irritant, and can cause burns

Principle: The phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then the nitrated tyrosine complexes mercury ions in the solution to form a brick-red , appearance of red color is positive test. Note: all phenols (compound having benzene ring and OH attached to it) give positive results in Millon’s test.

Procedure • To 2 ml of protein solution in a test tube, add 3 drops of Millon’s reagent. • Mix well and heat directly on a small flame. BWB 5 min • A white ppt is formed with albumin and casein (but not gelatin); • the ppt gradually turns into brick red.

Sakaguchi Test. • Objective: detection of amino acid containing gauanidium group. In other words it’s a test for, arginine. • Principle In alkaline solution, arginine react with αnaphthol and sodium hypobromite /chlorite as an oxidize agent, to form red complexes as a positive result.

• Add 1 ml of 3 N Na. OH solution to 1 ml of the protein solution, followed by addition of 0. 5 ml of 0. 1 % α- naphthol solution, and a few drops of 2 % sodium hypobromite solution (Na. OBr). • 2. The formation of a red color indicates the presence of a guanidinium group in the compound under examination.

Lead Sulfite Test • Objective: • This test specific for–SH [sulfhydral group ] containing amino acid (Cysteine). • Principle: • - Sulfur in cystine, is converted to sodium sulfide by boiling with 10% Na. OH. • - The Na 2 S can be detected by the precipitation of Pb. S (lead sulfide) from an alkaline solution. • when adding lead acetate Pb (CH 3 COO)2.

Methionine and cysteine contains sulfur group

Procedure 1. Place 1 ml of 2% casein, 2% egg albumin, 2% peptone, 2% gelatine and 0. 1 M cysteine into separate, labeled test tubes. 2. Add 2 ml of 10 % aqueous sodium hydroxide. Add 5 drops of 10 % lead acetate solution. 3. Stopper the tubes and shake them. Remove the stoppers and heat in a boiling water bath for 5 minutes. Cool and record the results.

Biuret test v Biuret structure: it is result of condensation of two molecule of urea • Principle: • The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in proteins to form a blue to violet complex known as the “biuret complex”. • This color change is dependent on the number of peptide bonds in the solution, so the more protein, the more intense the change.

• The Na. OH is there to raise the p. H of the solution to alkaline levels; the crucial component is the copper II ion (Cu 2+) from the Cu. SO 4. • When peptide bonds are present in this alkaline solution, the Cu 2+ions will form a coordination complex with 4 nitrogen atoms from peptide bonds. • N. B. Two peptide bonds at least are required for the formation of this complex.

A chelate is a chemical compound composed of a metal ion and a chelating agent. A chelating agent is a substance whose molecules can form several bonds to a single metal ion. In other words, a chelating agent is a multidentate ligand.

Procedure • To 2 ml of protein solution in a test tube, add 4 ml of reagent incubation 30 min • Result :

Observations Interpretation No change ( solution remains blue ) The solution turns from blue to violet( purple) The solution turns from blue to pink Proteins are not present Proteins are present Peptides are present ( Peptides or peptones are short chains of amino acid residues)

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